Brain-derived neurotrophic factor stimulates the retrograde pathway for axonal autophagy.

Autophagy is a lysosomal degradation pathway important for neuronal development, function, and survival. How autophagy in axons is regulated by neurotrophins to impact neuronal viability and function is poorly understood. Here, we use live-cell imaging in primary neurons to investigate the regulation of axonal autophagy by the neurotrophin brain-derived neurotrophic factor (BDNF) and elucidate whether autophagosomes carry BDNF-mediated signaling information. We find that BDNF induces autophagic flux in primary neurons by stimulating the retrograde pathway for autophagy in axons. We observed an increase in autophagosome density and retrograde flux in axons, and a corresponding increase in autophagosome density in the soma. However, we find little evidence of autophagosomes comigrating with BDNF. In contrast, BDNF effectively engages its cognate receptor TrkB to undergo retrograde transport in the axon. These compartments, however, are distinct from LC3-positive autophagic organelles in the axon. Together, we find that BDNF stimulates autophagy in the axon, but retrograde autophagosomes do not appear to carry BDNF cargo. Thus, autophagosomes likely do not play a major role in relaying neurotrophic signaling information across the axon in the form of active BDNF/TrkB complexes. Rather, BDNF likely stimulates autophagy as a consequence of BDNF-induced processes that require canonical roles for autophagy in degradation.

Retrograde Axonal Autophagy and Endocytic Pathways Are Parallel and Separate in Neurons.

Autophagy and endocytic trafficking are two key pathways that regulate the composition and integrity of the neuronal proteome. Alterations in these pathways are sufficient to cause neurodevelopmental and neurodegenerative disorders. Thus, defining how autophagy and endocytic pathways are organized in neurons remains a key area of investigation. These pathways share many features and converge on lysosomes for cargo degradation, but what remains unclear is the degree to which the identity of each pathway is preserved in each compartment of the neuron. Here, we elucidate the degree of intersection between autophagic and endocytic pathways in axons of primary mouse cortical neurons of both sexes. Using microfluidic chambers, we labeled newly-generated bulk endosomes and signaling endosomes in the distal axon, and systematically tracked their trajectories, molecular composition, and functional characteristics relative to autophagosomes. We find that newly-formed endosomes and autophagosomes both undergo retrograde transport in the axon, but as distinct organelle populations. Moreover, these pathways differ in their degree of acidification and association with molecular determinants of organelle maturation. These results suggest that the identity of autophagic and newly endocytosed organelles is preserved for the length of the axon. Lastly, we find that expression of a pathogenic form of α-synuclein, a protein enriched in presynaptic terminals, increases merging between autophagic and endocytic pathways. Thus, aberrant merging of these pathways may represent a mechanism contributing to neuronal dysfunction in Parkinson's disease (PD) and related α-synucleinopathies.SIGNIFICANCE STATEMENT Autophagy and endocytic trafficking are retrograde pathways in neuronal axons that fulfill critical degradative and signaling functions. These pathways share many features and converge on lysosomes for cargo degradation, but the extent to which the identity of each pathway is preserved in axons is unclear. We find that autophagosomes and endosomes formed in the distal axon undergo retrograde transport to the soma in parallel and separate pathways. These pathways also have distinct maturation profiles along the mid-axon, further highlighting differences in the potential fate of transported cargo. Strikingly, expression of a pathogenic variant of α-synuclein increases merging between autophagic and endocytic pathways, suggesting that mis-sorting of axonal cargo may contribute to neuronal dysfunction in Parkinson's disease (PD) and related α-synucleinopathies.